A distinct pattern of inflammatory lesions, consisting of focal non-suppurative necrotizing encephalitis, non-suppurative myocarditis and myositis have been observed in many aborted bovine fetuses submitted for diagnosis. The pattern of lesions, particularly in the brain, is similar to those seen with Toxoplasma gondii infections in sheep. However, cattle have been reported to be resistant to T. gondii infection (Dubey, Vet. Parasit. 22:177-202 (1986)). In 1988, a cyst-forming protozoal parasite was first identified by histopathological examination in fetuses (Barr, et al., Vet. Parasit. 27:354-61 (1990)). This parasite was morphologically similar to Toxoplasma, except that some of the cysts had thick walls, which was more similar to the Neospora caninum-like protozoan observed by Thilsted and Dubey (J. Vet. Diagnos. Invest. 1:205-9 (1989)) in aborted fetuses from a dairy in New Mexico.
Further studies showed the protozoal parasites associated with inflammatory lesions in aborted fetuses and neonatal calves in California had ultrastructural and antigenic features that were most similar to N. caninum parasites which were originally isolated from dogs (Dubey, et al., JAVMA 193:1259-63 (1988)). However, differences in the antigenic reactivity of the bovine protozoan and N. caninum when tested with a panel of antisera indicated they might not be from the same species (Barr, et al., Vet. Pathol. 28:110-16 (1991)).
A more complete understanding of the identity and biology of these bovine protozoa requires establishing continuous in vitro cultures of the parasites. Such cultures would also be valuable in the development of diagnostic assays and pharmaceutical compositions for the treatment and prevention of Neospora infections. The present invention addresses these and other needs.
In order to limit the spread of neosporosis, the development of an improved diagnostic test and a better understanding of the basic biology of the parasite are required. This invention contributes to this understanding by providing two antigens, N54 and N57, which can be used in a recombinant antigen-based ELISA. When sera from cattle of known disease status were tested (as will be described in more detail below) on the N54-based ELISA, N57-based ELISA, and the tachyzoite lysate-based ELISA, the recombinant antigen-based ELISAs had higher sensitivities and higher or similar specificities compared to the conventional lysate-based ELISA. Thus, the recombinant antigen-based ELISAs can be used for the serodiagnosis of bovine neosporosis.
In addition, this invention provides for NC-p65, which contains the N54 cDNA fragment. The discovery of this full-length cDNA, the encoded protein as well as its function allows the development of vaccines and therapeutics that will help to eliminate this devastating disease of cattle.
Specifically, this invention provides methods of detecting the presence of antibodies specifically immunoreactive with a bovine Neospora antigen in a biological sample (e.g., bovine serum). The method comprises contacting the sample with the Neospora antigen, thereby forming an antigen/antibody complex, and detecting the presence or absence of the complex. The Neospora antigen is typically an isolated recombinantly produced immunodominant Neospora antigen. In some embodiments, the antigen is immobilized on a solid surface and the complex is detected using a fluorescently labeled anti-bovine antibody.
The invention further provides methods of detecting the presence of Neospora in a biological sample. These methods comprise contacting the sample with an antibody specifically immunoreactive with a Neospora antigen, thereby forming an antigen/antibody complex, and detecting the presence or absence of the complex. The antibody (e.g., a monoclonal antibody) may be immobilized on a solid surface and the complex detected using a second labeled antibody. Typically, the biological sample is bovine fetal neurological tissue.
The methods of the invention also include detecting the presence of Neospora-specific nucleic acids in a biological sample by contacting the sample with an oligonucleotide probe which specifically hybridizes with a target Neospora-specific polynucleotide sequence and detecting the presence or absence of hybridization complexes. The methods may further comprise amplifying the target Neospora-specific polynucleotide sequence.
The invention further provides for pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an immunogenically effective amount of a bovine Neospora antigen, such as a recombinantly produced bovine Neospora polypeptide.
The pharmaceutical compositions are used in protecting a bovine animal from infection by bovine Neospora. The compositions are preferably administered to a cow or heifer when the animal is breeding. The pharmaceutical composition is usually administered parenterally.